Project Summary/Abstract Many human diseases result from improper regulation of cell growth (an increase in cell mass and size), proliferation, migration, survival and death. These processes are critically regulated by a complex containing the mammalian target of rapamycin (mTOR), Lst8 and Raptor, called the mTORC1 complex. The S6 protein kinases (S6K) are major effectors of mTORC1. The mTORC1/S6K signaling system is the cell's central integration point for multiple homeostatic inputs, sensing growth factor availability, energy levels, and amino acid sufficiency. Hyperactivation of mTORC1/S6K signaling is a common feature of nearly all human cancers. mTORC1 inhibitors, such as rapamycin and its analogs, are currently being clinically evaluated for the treatment of cancer. While the inhibitors have exhibited some promise, rapamycin- insensitive mTOR signaling also influences tumorigenesis, and feedback loops exist that up- regulate survival pathways following rapamycin treatment. Thus, additional therapeutic agents targeting other components of this pathway are needed. By taking a systems-wide approach towards defining mTORC1/S6K pathway regulation and the mechanisms by which this signaling system modulates various biological processes, we hope to provide insights that will lead to the identification of novel therapeutic strategies for the treatment of mTORC1/S6K- dependent cancers and other metabolic disorders. Aim 1 will focus on the connection between S6K1 signaling, gene expression and cell growth control through an S6K1-specific interacting protein SKAR. Approaches are described that investigate the role of SKAR and S6K1 in the regulation of mRNA biogenesis and protein translation. This aim also sets the foundation for how we will approach all mRNA binding proteins linked to the mTORC1/S6K signaling system identified in Aim 2. The approach outlined in Aim 2 combines a variety of biochemical purification approaches with mass spectrometry analysis to identify and validate a common set of proximal upstream regulators and downstream effectors of the S6K signaling system. The use of multiple converging lines of investigation will focus our efforts on the most critical components of the pathway, allowing us to dissect how S6K regulates so many disparate cellular processes. Aim 3 utilizes RNAi-based genetic approaches to elucidate the mechanism of homeostatic regulation of the mTORC1/S6K pathway. To this end, we have developed a sensitive, high- throughput, image-based screening strategy for monitoring S6K activity in vivo. We propose to utilize this unique assay to broadly interrogate mitogen- and nutrient-regulated inputs into the mTORC1/S6K pathway. Project Narrative. We hope that the studies outlined in this proposal will deepen our understanding of the defects in mTORC1/S6K signaling that are responsible for cancer progression and cell growth-associated diseases, such as the childhood cancer predisposition syndrome Tuberous Sclerosis. These studies will also impact our understanding of other metabolic diseases linked to S6K, such as diabetes and obesity. We believe such mechanistic insight will open the door to the identification of novel therapeutic strategies for inhibiting the growth factor and/or amino acid sensing arms of the mTORC1/S6K signaling network.